Fig. 7: Schematic model of generation of jawbone-like organoids.

mdEM and jawbone-like organoids were generated from human iPSCs in a stepwise manner, guided by signalling environments during embryogenesis, as depicted in the diagram. Initial SB/BMP4 treatment for 1 day, followed by SB/CHIR treatment, effectively induced HOX-negative NCC aggregates from iPSC aggregates (days 0–5). Transient RA treatment induced HOX-positive posterior NCC aggregates. From days 5 to 9, the combination of FGF8, EDN1 and BMP4 directed HOX-negative NCC aggregates to mdEM, recapitulating the proximal–distal patterning of embryonic mdEM. The regional identity of mdEM or mxEM was modulated by adjusting EDN1 signal activity. HOX-positive NCC aggregates did not differentiate into mdEM, underscoring the importance of initial NCC positional identity. mdEM exhibited later-stage regional patterning in response to exogenous mandibular epithelial signals (FGF8, SAG, EDN1 and BMP4), bifurcating into distal cap and proximal-oral molar domain. From days 9 to 38, under osteogenic conditions, mdEM formed jawbone-like organoids consisting of osteoblasts and network-forming osteocytes embedded in self-produced mineralized bone matrices. PA1-3, pharyngeal arch 1-3; mx, maxillary prominence; md, mandibular prominence; mdEM, md ectomesenchyme; SB, SB431542 (TGF-β inhibitor); CHIR, CHIR99021 (GSK3b inhibitor); BQ-123 (Endothelin Receptor Type A (EDNRA) antagonist); LDN, LDN193189 (BMP inhibitor); SAG (Hedgehog signalling agonist).